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  • Structural studies of Chikungunya virus maturation Moh Lan Yapa,b, Thomas Klosea, Akane Urakamic, S. Saif Hasana, Wataru Akahatac, and Michael G. Rossmanna,1

    aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; bDepartment of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Perak, Malaysia; and cVLP Therapeutics, Gaithersburg, MD 20878

    Edited by Robert M. Stroud, University of California, San Francisco, California, and approved November 10, 2017 (received for review July 25, 2017)

    Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphavi- ruses. Here we present a cryo-EM, 6.8-Å resolution structure of an “immature” Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less free- dom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protect- ing a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.

    alphavirus | Chikungunya virus | maturation | cryo-electron microscopy | conformational changes

    Chikungunya virus (CHIKV) is a mosquito-borne virus, whichwas first reported in Tanzania in 1952 (1) and later emerged as an epidemic in the French Reunion Island in 2005 (2). In the past decade, CHIKV has spread to more than 40 countries across Africa, Asia, and Europe, causing over a million infections in the Americas alone since 2014 (3). Among the symptoms of the disease are rash, myalgia, high fever, and, typically, severe arthritis (4). CHIKV is a member of the alphavirus genus in the Toga-

    viridae family (5). Other closely related and well-studied alphaviruses are Semliki Forest virus (SFV), Ross River virus (RRV), Sindbis virus (SINV), and Venezuelan Equine En- cephalitis virus (VEEV). Alphaviruses are spherical enveloped viruses with an ∼700-Å diameter and a T = 4 quasi-icosahedral symmetry. The genome of alphaviruses is an ∼12-kb positive- sensed single-stranded RNA molecule encoding four non- structural proteins (nsP1–4), which are required for virus rep- lication, and five structural proteins (capsid protein C, glycoproteins E1, E2, E3, and 6K) (6). The structural proteins are synthesized as a long polyprotein, which is then post- translationally cleaved into C, E1, 6K, and p62. A total of 240 copies of the C protein associate with a newly synthesized genomic RNA molecule to form a nucleocapsid in the host cell’s cytoplasm (7). The glycoproteins E1 and p62 interact to form heterodimers that subsequently trimerize into a viral spike in the endoplasmic reticulum (ER). The glycoprotein p62 is then cleaved into E2 and E3 by cellular furin during its trans- portation from the acidic environment of the Golgi and early endosomes to the neutral pH environment of the cell surface, releasing E3 (Movie S1). Virus budding occurs at the cell membrane where the nucleocapsid is enveloped by the glyco- proteins E1–E2 on the plasma lipid membrane. The protein 6K facilitates particle morphogenesis (8–10), but its position in the particle remains to be verified. Alpha- and flaviviruses (11) have many similarities. Their

    glycoprotein exteriors have icosahedral symmetry and surround a lipid membrane that, in turn, surrounds their RNA genome, which is associated with the capsid protein. A major difference between alpha- (12) and flaviviruses (13) is the maturation

    process. Flaviviruses are assembled as “immature” noninfectious particles in the ER of the host cell that are then proteolytically modified to produce infectious viruses on leaving the host cell. However, alphavirus components are proteolytically modified before assembly into mature viruses on the plasma membrane. In addition, a regular, icosahedral capsid shell is observed only in alphaviruses. During infection, a conserved sequence on the N-terminal regions of the capsid proteins binds to the host cell’s 60S ribosomal subunits, initiating the dissociation of the nu- cleocapsid and the release of the RNA from the nucleocapsid (14). This ribosome-binding site (RBS) is buried during nu- cleocapsid assembly but is exposed at the end of the maturation process (15, 16). In alphaviruses, there are 20 trimeric spikes located on the

    icosahedral threefold axes and another 60 trimeric spikes in general positions that obey T = 4 quasi-symmetry (17–19). Glycoprotein E1 is involved in cell fusion (20), and glycoprotein E2 interacts with host receptors (21) whereas glycoprotein E3 facilitates E1-p62 heterodimerization and prevents the ex- posure of the E1 fusion loops from premature fusogenic acti- vation (22, 23). Cryo-EM studies have shown that E3 remains associated with the mature virus of SFV (24), RRV (18), and VEEV (25). However, SINV (26, 27) and CHIKV (28) release E3 after budding.

    Significance

    Chikungunya virus (CHIKV) belongs to the alphavirus family, the members of which have enveloped icosahedral capsids. The maturation process of alphaviruses involves proteolysis of some of the structural proteins before assembling with nucleocapsids to produce mature virions. We mutated the proteolytic cleavage site on E2 envelope protein, which is necessary in initiating the maturation process. Noninfectious virus-like particles (VLP) equivalent to “immature” fusion incompetent particles were produced to study the immature conformation of CHIKV. We describe the 6.8-Å resolution electron microscopy structure of “immature” CHIK VLPs. Structural differences between the ma- ture and immature VLPs show that posttranslational processing of the envelope proteins and nucleocapsid is necessary to allow exposure of the fusion loop on glycoprotein E1 to produce an infectious virus.

    Author contributions: M.L.Y. and M.G.R. designed research; M.L.Y., T.K., A.U., and W.A. performed research; M.L.Y. and S.S.H. analyzed data; and M.L.Y. and M.G.R. wrote the paper.

    Conflict of interest statement: M.L.Y., T.K., S.S.H., and M.G.R. declare no competing fi- nancial interests. A.U. is an employee of VLP Therapeutics, and W.A. is an officer and shareholder of VLP Therapeutics.

    This article is a PNAS Direct Submission.

    Published under the PNAS license.

    Data deposition: The final immature Chikungunya VLP electron density map was depos- ited in the Electron Microscopy Data Bank, https://www.emdatabank.org (accession code EMD-8734), and structure coordinates have been deposited in the Protein Data Bank, www.rcsb.org/pdb (PDB ID code 5VU2). 1To whom correspondence should be addressed. Email: mr@purdue.edu.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1713166114/-/DCSupplemental.

    www.pnas.org/cgi/doi/10.1073/pnas.1713166114 PNAS | December 26, 2017 | vol. 114 | no. 52 | 13703–13707

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    http://movie-usa.glencoesoftware.com/video/10.1073/pnas.1713166114/video-1 http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1713166114&domain=pdf http://www.pnas.org/site/aboutpnas/licenses.xhtml https://www.emdatabank.org http://www.ebi.ac.uk/pdbe/entry/EMD-8734 http://www.rcsb.org/pdb http://www.rcsb.org/pdb/explore/explore.do?structureId=5VU2 mailto:mr@purdue.edu http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713166114/-/DCSupplemental http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1713166114/-/DCSupplemental www.pnas.org/cgi/doi/10.1073/pnas.1713166114

  • Here, we report the structure of immature CHIKV, which was determined using virus-like particles (VLPs) with mutations at the furin cleavage site on p62. The E3 remained associated with the E2, mimicking the precursor p62 in its immature confor- mation. A crystal structure of the E1-p62 heterodimer [Protein Data Bank (PDB) ID code 3N40 (29)] was fitted into the cryo- EM electron density map of immature CHIKV VLPs to examine the interactions of E1 and p62 with each other in the immature virus. A previous report showed that alphaviruses can be as- sembled in a partially mature, replication-competent state (25). Hence, the structure described here represents an intermediate structure of CHIKV during the assembly and maturation pro- cess. We showed that there are significant conformational dif- ferences between the mature and immature viruses, including the nucleocapsid, the transmembrane helices, and the cellular at- tachment sites on E2. The presence of E3 in the immature virus stabilized domain B on E2, protecting the fusion peptide on E1 from becoming exposed and fusogenic.

    Results and Discussion Cryo-EM Structure of Immature CHIKV.The cryo-EM density map of immature CHIK VLPs attained a 6.8-Å resolution (Fig. 1A). The virions had a diameter of 660 Å and, like mature virions, have T = 4 icosahedral symmetry. Central cross-sections of the re- construction showed that the immature virion (Fig. 1C) has a nucleocapsid, enveloped by a plasma membrane and an out- ermost layer of glycoproteins. Unlike flaviruses, alphaviruses,

    including CHIKV, have a well-ordered icosahedral nucleocapsid within the membrane envelope (Fig. 1B). Immature CHIKV virions, like mature CHIKV virions, have